Spleen samples from in vivo experiments were mashed, filtered through a 40-μm cell strainer, incubated in erythrocyte lysis buffer for 3 min at room temperature, filtered again, and lastly resuspended in PBS. Tumors were cut into small pieces and resuspended in culture medium containing collagenase type 2 (1 mg/ml; Worthington Biochemical) and deoxyribonuclease I (0.1 mg/ml; Boehringer Mannheim), incubated at 37°C for 1 hour, and then filtered through a 40-μm mesh. Samples were centrifuged, washed twice with PBS, and stained for 30 min at room temperature. A list of antibodies used is detailed in table S1. For coculture separation confirmation, cell suspensions were stained with a CD11b-FITC marker to identify the macrophage subpopulation. Gate settings, including those used for the gating of live single cells in an FSC-A versus FSC-H plot, were designed on the basis of fluorescence minus one controls and anti-rat/hamster compensation beads (BD Bioscience). TAMs were obtained using a FACSAria cell sorter (BD Biosciences). Data were acquired on a LSRFortessa X-20 or a FACSymphony flow cytometer (BD Biosciences) and analyzed with FlowJo (Tree Star Inc.).

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